The overall goal of this K01 award is to extend the scientific training and capabilities of the candidate and prepare him for an independent research career. This will be accomplished in the context of a detailed study of human growth hormone gene (hGH) expression. Prior studies from this laboratory have identified a tissue-specific locus control region (hGH LCR) that regulates the human growth hormone (hGH) gene cluster. The components of the hGH LCR are located quite far (15-32 kb) 5' to the target hGH-N gene promoter. Thus a central question in this model system is how the implied long-range gene activation between the hGH LCR and hGH-N promoter is mediated. During the current K01 mentored training period, the candidate has discovered that the hGH LCR is itself actively transcribed by Pol II and that the regions that are transcribed are also modified by specific and distinct patterns of histone alterations. The candidate's goal in the current proposal is to determine how these epigenetic alterations in LCR structure contribute to higher-order chromatin interactions that eventuate in hGH gene activation. The Hypothesis of the proposal is that the hGH LCR acts by recruitment and spreading of epigenetic modifiers. These modifications facilitate direct interactions within the LCR and between the LCR and the remote hGH-N promoter and they mediate relocation of the hGH locus to specific, transcriptionally-active sub-regions of the somatotrope nucleus. Aim I is to define the long-range interactions between the hGH LCR and hGH-N gene in somatotrope chromatin. This aim will address direct interactions within the LCR and between the LCR and the hGH-N promoter by applying Chromatin Conformation Capture (3C) analysis. The chromatin to be studied will be isolated from pituitaries and control tissues of mouse lines carrying informative transgenes. Aim II will establish functional relationships between hGH LCR activities and sub-nuclear localization of the hGH locus in somatotrope nuclei using high definition microscopic technology. A new collaboration will provide the candidate with additional training in the technology necessary to successfully implement this second aim. These studies address fundamental mechanisms of gene control and contribute to a foundation of knowledge necessary for understanding normal and pathological states of pituitary hormone expression. Such understanding establishes a basis for advances in diagnosis and treatment of endocrine disorders.